Nasal mucosa and blood cell transcriptome profiles do not reflect respiratory symptoms associated with moisture damage.

Upper and lower respiratory symptoms and asthma are adverse health effects associated with moisture-damaged buildings. Quantitative measures to detect adverse health effects related to exposure to dampness and mold are needed. Here, we investigate differences in gene expression between occupants of moisture-damaged and reference buildings. Moisture-damaged (N = 11) and control (N = 5) buildings were evaluated for dampness and mold by trained inspectors. The transcriptomics cohort consisted of nasal brushings and peripheral blood mononuclear cells (PBMCs) from 86 teachers, with/without self-perceived respiratory symptoms. Subject categories comprised reference (R) and damaged (D) buildings with (S) or without (NS) symptoms, that is, R-S, R-NS, DS, and D-NS. Component analyses and k-means clustering of transcriptome profiles did not distinguish building status (R/D) or presence of respiratory symptoms (S/NS). Only one nasal mucosa gene (YBX3P1) exhibited a significant change in expression between D-S and D-NS. Nine other nasal mucosa genes were differentially expressed between R-S and D-S teachers. No differentially expressed genes were identified in PBMCs. We conclude that the observed mRNA differences provide very weak biological evidence for adverse health effects associated with subject occupancy of the specified moisture-damaged buildings. This emphasizes the need to evaluate all potential factors (including those not related to toxicity) influencing perceived/self-reported ill health in moisture-damaged buildings.

Microbial characteristics in homes of asthmatic and non-asthmatic adults in the ECRHS cohort.

Microbial exposures in homes of asthmatic adults have been rarely investigated; specificities and implications for respiratory health are not well understood. The objectives of this study were to investigate associations of microbial levels with asthma status, asthma symptoms, bronchial hyperresponsiveness (BHR), and atopy. Mattress dust samples of 199 asthmatics and 198 control subjects from 7 European countries participating in the European Community Respiratory Health Survey II study were analyzed for fungal and bacterial cell wall components and individual taxa. We observed trends for protective associations of higher levels of mostly bacterial markers. Increased levels of muramic acid, a cell wall component predominant in Gram-positive bacteria, tended to be inversely associated with asthma (OR's for different quartiles: II 0.71 [0.39-1.30], III 0.44 [0.23-0.82], and IV 0.60 [0.31-1.18] P for trend .07) and with asthma score (P for trend .06) and with atopy (P for trend .02). These associations were more pronounced in northern Europe. This study among adults across Europe supports a potential protective effect of Gram-positive bacteria in mattress dust and points out that this may be more pronounced in areas where microbial exposure levels are generally lower.

Microbial growth in building material samples and occupants' health in severely moisture-damaged homes.

There is no commonly approved approach to detect and quantify the health-relevant microbial exposure in moisture-damaged buildings. In 39 single-family homes with severe moisture damage, we studied whether concentrations of viable microbes in building material samples are associated with health among 71 adults and 68 children, and assessed with symptoms questionnaires, exhaled NO, and peak expiratory flow (PEF) variability. Symptoms were grouped into three scores: upper respiratory symptoms, lower respiratory symptoms, and general symptoms. The homes were divided into three groups based on viable counts of fungi, actinomycetes, and total bacteria cultivated from building material samples. Highest group of actinomycete counts was associated with more general symptoms, worse perceived health, and higher daily PEF variability (aOR 12.51; 1.10-141.90 as compared to the lowest group) among adults, and with an increase in lower respiratory symptoms in children, but the confidence intervals were wide. We observed significant associations of fungal counts and total microbial score with worse perceived health in adults. No associations with exhaled NO were observed.

Synergistic proinflammatory interactions of microbial toxins and structural components characteristic to moisture-damaged buildings.

Indoor exposure to microbes and their structural and metabolic compounds is notoriously complex. To study proinflammatory interactions between the multiple microbial agents, macrophages derived from human THP-1 monocytic cells were exposed to several concentrations of microbial toxins alone (emodin, enniatin B, physcion, sterigmatocystin, valinomycin) and in combination with microbial structural components (bacterial lipopolysaccharide [LPS] or fungal ß-glucan). While the expression of proinflammatory cytokines TNFα and IL-1ß to single toxins alone was modest, low-dose co-exposure with structural components increased the responses of emodin, enniatin B, and valinomycin synergistically, both at the mRNA and protein level, as measured by RT-qPCR and ELISA, respectively. Co-exposure of toxins and ß-glucan resulted in consistent synergistically increased expression of several inflammation-related genes, while some of the responses with LPS were also inhibitory. Co-exposure of toxins with either ß-glucan or LPS induced also mitochondrial damage and autophagocytosis. The results demonstrate that microbial toxins together with bacterial and fungal structural components characteristic to moisture-damaged buildings can have drastic synergistic proinflammatory interactions at low exposure levels.

School attendance and daily respiratory symptoms in children: influence of moisture damage.

We investigated the effect of weekends and school holidays on the daily frequency and severity of respiratory and other symptoms among children attending schools with (index) or without (reference) moisture damage in Spain, the Netherlands, and Finland. Throughout 1 year, parents of 419 children with a respiratory condition attending index (n=15) or reference (n=10) primary schools completed three symptom diaries. We assessed associations between lower respiratory tract, upper respiratory tract or allergy, and other symptom scores and school day, weekend, or summer holiday using mixed regression models stratified by country and moisture damage. We evaluated interactions between moisture damage and type of day. We combined country-specific estimates (incidence rate ratios [IRRs] and 95% confidence interval [CI]) in meta-analyses. Symptom scores were lower during weekends and holiday. Lower respiratory tract symptoms were statistically significantly less common during holiday with strongest effect in index schools (IRR=0.7; CI=0.6-0.8). Reporting of other symptoms was more reduced during holiday in index (IRR=0.6; CI=0.4-0.9) than in reference (IRR=0.95; CI=0.8-1.2) schools (interaction P<.01). In conclusion, symptoms were less frequent and/or severe during summer holiday and weekends. This pattern was stronger among children attending moisture-damaged schools, suggesting potential improvement in moisture damage-related symptoms during school breaks.

Inflammatory potential in relation to the microbial content of settled dust samples collected from moisture-damaged and reference schools: results of HITEA study.

Aiming to identify factors causing the adverse health effects associated with moisture-damaged indoor environments, we analyzed immunotoxicological potential of settled dust from moisture-damaged and reference schools in relation to their microbiological composition. Mouse RAW264.7 macrophages were exposed to settled dust samples (n = 25) collected from moisture-damaged and reference schools in Spain, the Netherlands, and Finland. After exposure, we analyzed production of inflammatory markers [nitric oxide (NO), tumor necrosis factor-α (TNF-)α, interleukin (IL)-6, and macrophage inflammatory protein (MIP)2] as well as mitochondrial activity, viability, apoptosis, and cell cycle arrest. Furthermore, particle counts, concentration of selected microbial groups as well as chemical markers such as ergosterol, 3-hydroxy fatty acids, muramic acid, endotoxins, and glucans were measured as markers of exposure. Dust from moisture-damaged schools in Spain and the Netherlands induced stronger immunotoxicological responses compared to samples from reference schools; the responses to Finnish samples were generally lower with no difference between the schools. In multivariate analysis, IL-6 and apoptosis responses were most strongly associated with moisture status of the school. The measured responses correlated with several microbial markers and numbers of particles, but the most important predictor of the immunotoxicological potential of settled dust was muramic acid concentration, a marker of Gram-positive bacteria.

Microbial secondary metabolites in homes in association with moisture damage and asthma.

We aimed to characterize the presence of microbial secondary metabolites in homes and their association with moisture damage, mold, and asthma development. Living room floor dust was analyzed by LC-MS/MS for 333 secondary metabolites from 93 homes of 1-year-old children. Moisture damage was present in 15 living rooms. At 6 years, 8 children had active and 15 lifetime doctor-diagnosed asthma. The median number of different metabolites per house was 17 (range 8-29) and median sum load 65 (4-865) ng/m(2) . Overall 42 different metabolites were detected. The number of metabolites present tended to be higher in homes with mold odor or moisture damage. The higher sum loads and number of metabolites with loads over 10 ng/m(2) were associated with lower prevalence of active asthma at 6 years (aOR 0.06 (95% CI <0.001-0.96) and 0.05 (<0.001-0.56), respectively). None of the individual metabolites, which presence tended (P < 0.2) to be increased by moisture damage or mold, were associated with increased risk of asthma. Microbial secondary metabolites are ubiquitously present in home floor dust. Moisture damage and mold tend to increase their numbers and amount. There was no evidence indicating that the secondary metabolites determined would explain the association between moisture damage, mold, and the development of asthma.

Indoor fungi: companions and contaminants.

This review discusses the role of fungi and fungal products in indoor environments, especially as agents of human exposure. Fungi are present everywhere, and knowledge for indoor environments is extensive on their occurrence and ecology, concentrations, and determinants. Problems of dampness and mold have dominated the discussion on indoor fungi. However, the role of fungi in human health is still not well understood. In this review, we take a look back to integrate what cultivation-based research has taught us alongside more recent work with cultivation-independent techniques. We attempt to summarize what is known today and to point out where more data is needed for risk assessment associated with indoor fungal exposures. New data have demonstrated qualitative and quantitative richness of fungal material inside and outside buildings. Research on mycotoxins shows that just as microbes are everywhere in our indoor environments, so too are their metabolic products. Assessment of fungal exposures is notoriously challenging due to the numerous factors that contribute to the variation of fungal concentrations in indoor environments. We also may have to acknowledge and incorporate into our understanding the complexity of interactions between multiple biological agents in assessing their effects on human health and well-being.

Determinants, reproducibility, and seasonal variation of bacterial cell wall components and viable counts in house dust.

The objectives of this study were (i) to assess the determinants that affect concentrations of the bacterial cell wall components 3-hydroxy fatty acids (3-OH FAs) and muramic acid and of total viable bacteria and actinomycetes in house dust; and (ii) to examine the seasonal variation and reproducibility of these bacterial cell wall components in house dust. A number of lifestyle and environmental factors, mostly not consistent for different bacterial measures but commonly including the type of dwelling and farming (number of livestock), explained up to 37% of the variation of the bacterial concentrations in 212 homes in Eastern Finland. The reproducibility of 3-OH FAs and muramic acid measurements in house dust were studied in five urban homes and were found to be generally high (ICC 74-84%). Temporal variation observed in repeated sampling of the same home throughout a year was more pronounced for 3-OH FAs determinations (ICC 22%) than for muramic acid (ICC 55-66%). We conclude that determinants vary largely for different types of bacterial measurements in house dust; the measured parameters represent different aspects of the bacterial content indoors. More than one sample is needed to describe bacterial concentrations in house dust in the home environment due to large temporal variation.

Determinants, reproducibility, and seasonal variation of ergosterol levels in house dust.

UNLABELLED: This study aimed to clarify the determinants that affect the concentrations of ergosterol and viable fungi in house dust and to examine the seasonal variation and reproducibility of ergosterol concentrations indoors. In studying the determinants, dust samples from living room floors and vacuum cleaner dust bags were collected from 107 farming and 105 non-farming homes. Ergosterol levels were determined with gas chromatography-mass spectrometry,and the dust bag dust was cultivated for enumeration of fungal genera. Lifestyle and environmental factors, for example using of the fireplace, and visible mold observations in homes, explained 20­26% of the variation of fungal concentrations. For the reproducibility study, samples were collected from five urban homes in four different seasons. The reproducibility of ergosterol determinations within a sample was excellent (ICC = 89.8) for floor dust and moderate (ICC = 63.8) for dust bag dust, but poor when sampling the same home throughout a year (ICC = 31.3 and 12.6, respectively) due to large temporal variation in ergosterol concentrations. In conclusion, environmental characteristics only partially predicted the variation of fungal concentrations. Based on these studies, we recommend repeated sampling of dust over time if one seeks to adequately describe overall fungal levels and exposure in a home. PRACTICAL IMPLICATIONS: This study shows that levels of ergosterol and viable fungi in house dust are related to visible mold observations. Only 20% of the variation in fungal levels can be explained with questionnaires, and therefore, environmental samples need to be taken in addition. Reproducibility of ergosterol determination was excellent for floor dust, and thus, ergosterol measurements from floor dust samples could be suitable for assessing the fungal load in building investigations. The temporal variation needs to be taken into account when describing the ergosterol concentration of urban homes.

Quantity and diversity of environmental microbial exposure and development of asthma: a birth cohort study.

BACKGROUND: Early-life exposure to environmental microbial agents may be associated with the development of allergies. The aim of the study was to identify better ways to characterize microbial exposure as a predictor of respiratory symptoms and allergies. METHODS: A birth cohort of 410 children was followed up until 6 years of age. Bacterial endotoxin, 3-hydroxy fatty acids, N-acetyl-muramic acid, fungal extracellular polysaccharides (EPS) from Penicillium and Aspergillus spp., ß-D-glucan, ergosterol, and bacterial or fungal quantitative polymerase chain reactions (qPCRs) were analyzed from dust samples collected at 2 months of age. Asthma, wheezing, cough, and atopic dermatitis were assessed using repeated questionnaires. Specific IgEs were determined at the age of 1 and 6 years. RESULTS: Only few associations were found between single microbial markers and the studied outcomes. In contrast, a score for the total quantity of microbial exposure, that is, sum of indicators for fungi (ergosterol), Gram-positive (muramic acid) bacteria, and Gram-negative (endotoxin) bacteria, was significantly (inverted-U shape) associated with asthma incidence (P < 0.001): the highest risk was found at medium levels (adjusted odds ratio (aOR) 2.24, 95% confidence interval (95% CI) 0.87-5.75 for 3rd quintile) and the lowest risk at the highest level (aOR 0.34, 95% CI 0.09-1.36 for 5th quintile). The microbial diversity score, that is, sum of detected qPCRs, was inversely associated with risk of wheezing and was significantly (inverted-U shape) associated with sensitization to inhalant allergens. CONCLUSION: Score for quantity of microbial exposure predicted asthma better than single microbial markers independently of microbial diversity and amount of dust. Better indicators of total quantity and diversity of microbial exposure are needed in studies on the development of asthma.

Endotoxin levels in settled airborne dust in European schools: the HITEA school study.

Indoor exposure to microbial agents is known to influence respiratory health. Besides home exposure, exposure in schools can affect respiratory health. In this study, we measured endotoxin in settled dust in primary schools in three European countries from three different geographical regions with different climates. Our aim was to characterize endotoxin levels in primary schools and evaluate associations with potential determinants. Endotoxin levels were repeatedly assessed in 23 schools in Spain (n = 7), the Netherlands (n = 10), and Finland (n = 6) using electrostatic dustfall collectors. In total, 645 measurements were taken in 237 classrooms. Endotoxin levels differed significantly between countries; Dutch schools had the highest levels, while Finnish schools showed the lowest levels. In each country, differences in endotoxin levels were observed between schools and over the sampling periods. Estimates improved after adjustment for sampling period. Factors affecting endotoxin levels in a school differed per country. In general, endotoxin levels were higher in lower grades and in classrooms with higher occupancy. School endotoxin levels may contribute significantly to total endotoxin exposure in children and teachers. As the correlation between the repeated measurements is reasonable, single endotoxin measurements form a reasonable basis for estimating annual endotoxin levels in schools.

Winter ventilation rates at primary schools: comparison between Portugal and Finland.

This study focused on examination of ventilation rates in classrooms with two different types of ventilation systems: natural and mechanical. Carbon dioxide (CO2) measurements were conducted in primary schools of Portugal characterized by natural ventilation and compared to Finland where mechanical ventilation is the norm. The winter period was selected since this season exerts a great influence in naturally ventilated classrooms, where opening of windows and doors occurs due to outdoor atmospheric conditions. The ventilation rates were calculated by monitoring CO2 concentrations generated by the occupants (used as a tracer gas) and application of the buildup phase method. A comparison between both countries' results was conducted with respect to ventilation rates and how these levels corresponded to national regulatory standards. Finnish primary schools (n = 2) registered a mean ventilation rate of 13.3 L/s per person, which is higher than the recommended ventilation standards. However, the Finnish classroom that presented the lowest ventilation rate (7.2 L/s per person) displayed short-term CO2 levels above 1200 ppm, which is the threshold limit value (TLV) recommended by national guidelines. The Portuguese classrooms (n = 2) showed low ventilation rates with mean values of 2.4 L/s per person, which is markedly lower than the minimum recommended value of 7 L/s per person as defined by ASHRAE and 20% less than the REHVA minimum of 3 L/s per person. Carbon dioxide levels of 1000 ppm, close to the TLV of 1200 ppm, were also reached in both Portuguese classrooms studied. The situation in Portugal indicates a potentially serious indoor air quality problem and strengthens the need for intervention to improve ventilation rates in naturally ventilated classrooms.

Occurrence of moisture problems in schools in three countries from different climatic regions of Europe based on questionnaires and building inspections - the HITEA study.

UNLABELLED: The aim of this study was to assess occurrence of dampness and mold in school buildings in three European countries (the Netherlands, Spain, and Finland), representing different climatic regions. An assessment was performed utilizing both questionnaires and on-site building investigations, and the agreement between these two methods was evaluated for validation purposes. On the basis of questionnaire data from a representative sample of schools, different types of moisture problems were reported in 24-47% of all school buildings at the time of the study. Most commonly reported was dampness in the Netherlands, moisture/water damage in Spain, and mold odor in Finland. Subsequently, 20-24 schools per country were selected for on-site inspections by trained staff. The overall agreement between the questionnaire and inspection data was good (kappa-value 0.62), however, with large differences (0.39-0.91) between countries. Extrapolating from the inspection data, the minimum estimates for prevalence of moisture problems in school buildings are 20% in the Netherlands, 41% in Spain, and 24% in Finland. In conclusion, moisture problems (such as moisture damage, dampness, and mold) are relatively common in schools. The occurrence and severity may vary across geographical areas, which can be partly explained by building characteristics. PRACTICAL IMPLICATIONS: On the basis of this study, the prevalence of verified moisture problems in school buildings was highest in Spain, but lower and similar in Finland and the Netherlands. Questionnaire-based surveys can be used to assess moisture problems in school buildings, but because of large variation in agreement with inspection data, the questionnaire needs to be validated by on-site inspections in a subsample of the surveyed buildings.

Geographical variation and the determinants of domestic endotoxin levels in mattress dust in Europe.

UNLABELLED: Endotoxin exposures have manifold effects on human health. The geographical variation and determinants of domestic endotoxin levels in Europe have not yet been extensively described. To investigate the geographical variation and determinants of domestic endotoxin concentrations in mattress dust in Europe using data collected in the European Community Respiratory Health Survey follow-up (ECRHS II). Endotoxin levels were measured in mattress dust from 974 ECRHS II participants from 22 study centers using an immunoassay. Information on demographic, lifestyle, and housing characteristics of the participants was obtained in face-to-face interviews. The median endotoxin concentration in mattress dust ranged from 772 endotoxin units per gram (EU/g) dust in Reykjavik, Iceland, to 4806 EU/g in Turin, Italy. High average outdoor summer temperature of study center, cat or dog keeping, a high household crowding index, and visible damp patches in the bedroom were significantly associated with a higher endotoxin concentrations in mattress dust. There is a large variability in domestic endotoxin levels across Europe. Average outdoor summer temperature of study center, which explains only 10% of the variation in domestic endotoxin level by center, is the strongest meteorological determinant. The observed variation needs to be taken into account when evaluating the health effects of endotoxin exposures in international contexts. PRACTICAL IMPLICATIONS: The incoherent observations of the health effects of endotoxin may be partly owing to the geographical heterogeneity of endotoxin exposure. Therefore, the observed variation should be considered in further studies. Measurements of indoor endotoxin are recommended as an indicator for the level of exposures of individual domestic environments.

Co-occurrence of toxic bacterial and fungal secondary metabolites in moisture-damaged indoor environments.

UNLABELLED: Toxic microbial secondary metabolites have been proposed to be related to adverse health effects observed in moisture-damaged buildings. Initial steps in assessing the actual risk include the characterization of the exposure. In our study, we applied a multi-analyte tandem mass spectrometry-based methodology on sample materials of severely moisture-damaged homes, aiming to qualitatively and quantitatively describe the variety of microbial metabolites occurring in building materials and different dust sample types. From 69 indoor samples, all were positive for at least one of the 186 analytes targeted and as many as 33 different microbial metabolites were found. For the first time, the presence of toxic bacterial metabolites and their co-occurrence with mycotoxins were shown for indoor samples. The bacterial compounds monactin, nonactin, staurosporin and valinomycin were exclusively detected in building materials from moist structures, while chloramphenicol was particularly prevalent in house dusts, including settled airborne dust. These bacterial metabolites are highly bioactive compounds produced by Streptomyces spp., a group of microbes that is considered a moisture damage indicator in indoor environments. We show that toxic bacterial metabolites need to be considered as being part of very complex and diverse microbial exposures in 'moldy' buildings. PRACTICAL IMPLICATIONS: Bacterial toxins co-occur with mycotoxins in moisture-damaged indoor environments. These compounds are measurable also in settled airborne dust, indicating that inhalation exposure takes place. In attempts to characterize exposures to microbial metabolites not only mycotoxins but also bacterial metabolites have to be targeted by the analytical methods applied. We recommend including analysis of samples of outdoor air in the course of future indoor assessments, in an effort to better understand the outdoor contribution to the indoor presence of microbial toxins. There is a need for a sound risk assessment concerning the exposure to indoor microbial toxins at concentrations detectable in moisture-damaged indoor environments.

Lactobacillus rhamnosus strain GG modulates intestinal absorption, fecal excretion, and toxicity of aflatoxin B(1) in rats.

In this study, the modulation of aflatoxin B(1) (AFB(1)) uptake in rats by administration of the probiotic Lactobacillus rhamnosus GG was demonstrated. Fecal AFB(1) excretion in GG-treated rats was increased via bacterial AFB(1) binding. Furthermore, AFB(1)-associated growth faltering and liver injury were alleviated with GG treatment.

Toxicity of ochratoxin A in aBrevibacillus brevis - Growth inhibition assay.

Ochratoxin A (OTA) is a nephrotoxic, carcinogenic and immunosuppressive mycotoxin. It can be detoxified by various microorganisms, e.g. different yeast strains, via metabolisation into ochratoxin α (OTα). Within this study a growth inhibition assay was developed to compare the toxicity of OTA and its degradation product OTα. As an indicator organismBrevibacillus brevis was used. The assay was performed in microtiterplates. Growth inhibition was determined by comparing the optical density values ofBrevibacillus brevis cultures grown in medium supplemented with OTA/OTα and OTA/OTα-free medium, respectively.It could be shown thatB. brevis is sensitive to OTA (EC100=0.5 mg/L±0.03 mg/L), which is not the case for its metabolite OTα. Therefore this bioassay is a useful tool to show the detoxification of OTA to OTα by microbial degradation.

Investigation of different yeast strains for the detoxification of ochratoxin A.

High concentrations of ochratoxin A (OTA) in feed lead to growth depression in animals. It has been reported that binders can be used for deactivating aflatoxins but not for other mycotoxins without negatively influencing the animals health. In this study a strain from the genus ofTrichosporon with the ability to cleave ochratoxin A very selectively into phenylalanine and the non-toxic ochratoxin α (OTα) could be isolated. This strain was selected from a pool of OTA detoxifying microorganism by carrying out several investigations.Trichosporon sp. nov. can be fermented and stabilized. In a feeding trial with broilers lyophilizedTrichosporon-cells could compensate performance losses caused by OTA.